Hemophilia has proven a fruitful model for the study of gene-based approaches to the treatment of disease, and it seems likely that such an approach will be developed for widespread application in the near future. A substantial proportion of patients with severe hemophilia, develop inhibitory antibodies to infused clotting factor, which they perceive as a "foreign" protein. These individuals fail to respond to clotting factor concentrates and until recently presented one of the most difficult management problems in hemophilia Experience over the past decade has shown that administration of recombinant F.VIIa in doses sufficient to achieve circulating levels of 2-4mug/ml levels can result in effective hemostasis in individuals with inhibitors. In this application we propose to develop a gene-based approach to administration of VIIa in hemophilic animals where inhibitor formation has been induced. Building on our success with AAV vectors administered to liver, we will develop AAV vectors that express an engineered F.VII construct that is cleaved to F.VIIa intracellular and secreted as the activated form. In aim 1, we will carry out short-term experiments to determine whether we can achieve hemostasis in a mouse model of hemophilic inhibitors by portal vein injection of an AAV-F.VIIa vector. In the second aim we will carry out long-term studies of clotting parameters in mice that continuously express F.VIIa at a series of defined levels. The purpose of these experiments is to determine whether there is any baseline level of F.VIIa expression that will result in improvement in clotting parameters without serious adverse effects. In the second part of this aim we will develop AAV-VIIa vectors controlled by a "switch" that can be activated by the drug doxycycline, and determine whether such a system can be used to reduce unwanted side effects associated with long-term expression of VIIa, yet still serve to prevent bleeding in response to a hemostatic challenge. In the third aim we will seek to extend these findings to dogs with hemophilia and inhibitors, and in the fourth aim we will assess the immunogenicity of these findings to dogs with hemophilia and inhibitors, and in the fourth aim we will assess the immunogenicity of the modified F.VII constructs used to generate fully processed F.VIIa as a secreted product. Successful completion of these pre-clinical studies should help to establish whether a gene-based approach to treatment of hemophilic inhibitors is feasible.